Supercritical fluid chromatography (SFC) is a separation technique similar to high performance liquid chromatography (HPLC), except one of the fluids used as a solvent is a highly compressible liquefied gas. Supercritical fluid extraction (SFE) is a related technique but with somewhat lower requirements for accurate flow or pressure control. The most common fluid used in SFC (and SFE) is carbon dioxide, which will be considered as representative of all such fluids.
At room temperature and atmospheric pressure, carbon dioxide is a low density gas (density approximately 0.002 g/cm3). The desirable characteristics of carbon dioxide for SFC and SFE are only achieved when the carbon dioxide is held at a liquid-like density, usually between 0.6 and 1.0 g-cm-3, by raising it's pressure to 80 to 600 Bar, while keeping the temperature in the general range of 20° to 100° C., and more commonly between 35 to 60° C. Under such conditions, the carbon dioxide: 1.) acts as a solvent, 2.) exhibits very high solute binary diffusion coefficients (allows higher flow rates than in HPLC), and 3.) exhibits very low viscosity (generates lower pressure drops across columns compared to HPLC).
To be useful in SFC (or SFE), the carbon dioxide is compressed to high pressures and pumped as a liquid or as a supercritical fluid, at a liquid like density, through a separation column. To prevent it from expanding to atmospheric pressure in the column, a back pressure regulator (BPR) is placed downstream of the separation column to keep the column outlet pressure above typically 80 Bar. Detectors capable of operating under high pressure may be mounted between the column and the BPR. Low pressure detectors may be mounted in the flow stream directly downstream of the BPR.
The pressure drops to near atmospheric pressure as it passes through the BPR. Both the BPR, and the fluid are cooled by the adiabatic expansion of the fluid. If pure carbon dioxide is used, it can actually form “dry ice” and intermittently plug the flow path, if external heat is not applied. Plugging and subsequent thawing results in intermittent, noisy flow, and loss of pressure/flow control. With modified fluids (where small amounts of other fluids are mixed with the carbon dioxide), “slush” of partially frozen carbon dioxide and modifier sometimes forms, causing the BPR to sputter, and loose pressure/flow control. Consequently, all BPR's used in SFC have been heated to maintain smooth flow control. Intermittent heating, such as performed by on-off heater controllers, can cause serious pressure instability. The heater control must be carefully optimized to avoid inducing pressure oscillations. With low levels of heating, the plugging problem can be avoided, but water from the atmosphere often condenses on the outlet line of the BPR. This liquid water, or sometimes ice, can cause multiple additional problems, and needs to be controlled/eliminated. Heating the BPR to a higher temperature eliminates the condensation/ice formation problem. However, the use of excessively high temperatures could damage thermally labile compounds passing through the BPR and need to be avoided.
In order to be appropriate for use in SFC, a BPR must be stable, accurate and repeatable, with appropriately low, unswept volume, and generate low UV detector noise. In addition it should be relatively inexpensive and easy to use and maintain. Users sometimes program pressure versus time, making electromechanical control desirable. As equipment has become more and more computer controlled, it has become desirable to have all the set points stored and downloaded from a single electronic method file, making electromechanical control of the BPR even more desirable.
Pressure affects retention and selectivity in SFC, although not very strongly, particularly when modified mobile phases are used. If the pressure drifts or wanders, retention times will drift and wander. Validating a method on a single instrument in a single lab requires reasonable pressure stability. In general, a series of injections must have retention time reproducibility less than +/−1%. Transferring methods from one lab to another requires reasonable pressure accuracy.
Standard mechanical back pressure regulators, such as those available from Tescom, generally have a large surface in direct contact with the fluid being controlled, which allows smoother, more precise control. However the internal volume of such devices is often very large, making them incompatible with some applications of SFC. Mechanical regulators often have as much as 5 mls of poorly swept internal volume. On the analytical scale, using 5 μm particles, on a 4.6 mm ID column, the volume containing a peak is roughly 75 to 200 μL. Any component in the flow path should have a dead volume roughly ⅕th these volumes or smaller, and the volume should be well swept, if the user expects to retain the separation (resolution between peaks) during decompression through the BPR. If the user wishes to place a detector downstream of the BPR, the volume of the BPR must be no larger than 10 to 40 μL, and preferably on the lowest end of this range. Unfortunately, the smaller the volume of the BPR the more difficult it is to control the pressure.
Mechanical BPR's, with large internal volumes, may be adequate for preparative and even semi-preparative scale SFC. On the preparative scale, samples are contained in 10's to 100's of mls of mobile phase. The BPR should have an inner volume less than ⅕th the volume containing the peak.
UV detectors are the most common detector type used in SFC. UV detector response is affected by refractive index changes in the mobile phase. The refractive index of carbon dioxide is highly dependent on temperature and pressure. In the past, the most common outlet pressure used in SFC has been 100 Bar. The most common column temperature used in SFC has been 40° C. At 40° C. the refractive index changes from 1.1120 at 90 Bar to 1.1606 at 110 Bar, a change of over 4%. This represents a change of approximately 0.2%/Bar. Oscillations in refractive index cause the light beam passing through a UV detector cell to be bent a variable amount. Pressure fluxuations generate refractive index fluxuations which causes variations in the light hitting the photosensitive portion of the detector. It is desirable to minimize pressure fluxuations to achieve low UV detector noise.
Prior back pressure regulator designs have proven to be temperamental in terms of both control and/or calibration for a specified range function. What is needed is device that dramatically reduces calibration requirements while maintaining dramatically improved control.